The present invention relates generally to materials and methods for use in immunological procedures for isolation and quantitative detection of colony stimulating factor, commonly known as "CSF", from biological fluids. More specifically, the invention relates to two monoclonal anti-CSF-1 antibodies, produced by two novel hybridoma cell lines (A.T.C.C. HB8207 and A.T.C.C. HB8208) and to uses of these antibodies in diagnostic assays of CSF-1 content on human and animal fluids and in procedures for isolating CSF-1 in quantity.
The cells of the immune system that respond to the presence of antigenic substances in the body originate from hematopoietic tissue, the bone marrow. Approximately one-half of the tissue in bone marrow is dedicated to hematopoiesis or blood cell formation. The development of blood cells arises from primitive undifferentiated stem cells and diverges into several distinct lines which include, among others, cells of the granulocytic and monocytic series (i.e., macrophages, monocytes, granulocytes).
Colony stimulating factors ("CSFs") are glycoprotein hemopoietic cell growth factors which, in semi-solid culture media, stimulate the growth and differentiation of hemopoietic precursor cells into colonies of granulocytes and/or macrophages. At least four separable subclasses of CSF have been identified, each exhibiting different physical properties and target cell specificities. The subclasses have been described by reference to the types of mature cells they stimulate production of in culture. One subclass stimulates macrophage production exclusively; a second stimulates neutrophilic granulocyte and macrophage production; a third stimulates neutrophilic granulocyte production; and a fourth stimulates eosinophilic granulocyte production.
CSF-1 is the most clearly delineated subclass of CSF. It stimulates the proliferation of cells of the mononuclear phagocytic series, (i.e., undifferentiated cells, blood mononuclear cells and macrophages) only. In the absence of CSF-1 undifferentiated mononuclear phagocytic cells rapidly become unresponsive or die, while macrophages survive, but in a non-proliferating state. In the presence of CSF-1, responsive cells have a finite proliferative capacity that decreases as the cells mature. Both cell types elevate their secretion of plasminogen activation in response to CSF-1. CSF-1 is the only subclass to be unambiguously defined by subclass specific radioimmunoassays and radioreceptor assays. Specific cell surface receptors which mediate the biological effects of this subclass occur exclusively on mononuclear phagocytic cells. CSF-1 has been purified from several murine sources, human lung tissue, human urine and human pancreatic tumor cells. It is a sialic acid glycoprotein with a molecular weight of 40,000-70,000.
Information concerning the structure-function relationships of CSF-1 has been limited due to difficulties involved in obtaining even microgram amounts of the material in purified form. CSF-1 comprises only about 0.1% of the total protein of the most suitable starting materials, so that its purification requires large starting volumes. Additionally, many primary tissue sources, because of contamination with serum, contain too large a quantity of heterogenous glycoproteins from which separation of CSF-1 is difficult. A presently more desirable source of CSF-1 is a human pancreatic carcinoma cell line, MIA-Pa-Ca-2 (A.T.C.C. CRL 1420), which is reported to secrete relatively large amounts of CSF-1 into serum-free conditioned medium. Wu, et al., J.Clin.Invest., 65, 772-775 (1980). A method for purification of CSF-1 to apparent homogeneity from the medium of the MIA-Pa-Ca-2 cells is described in Wu, et al., J.Biol.Chem., 254, 6226-6228 (1979). Other reports of studies on the structure, function and assay of CSF-1 are found in the following: Stanley, et al., J.Imm.Methods, 42, 253-284 (1981); Das, et al., Blood, 58, 630-641 (1981); Das, et al., J.Cellular Physiol., 104, 359-366 (1980); and Das, et al., J.Biol.Chem., 257, 13679-13684 (1982).
Pure human CSF-1 in quantity would provide a desirable reagent for biological studies on the mechanism of hormone action in the immune system and could be specifically applied in immunoassays, such as radioimmunoassays ("RIAs"), enzyme linked immunosorbent assays ("ELISAs"), and the like, for quantitative detection of CSF-1 in biological fluids such as blood, serum and urine. Highly purified CSF-1 may also have therapeutic potential in the treatment of cancer patients displaying decreased granulocyte production and/or reduced circulating peripheral granulocyte count resulting from chemotherapy. Inhibition of bone marrow precursor cells due to radiation exposure or chemotherapy puts the patient at a risk of infection by disabling the circulating cells of the immune system and, in fact, a decrease in blood granulocytes is often the principal factor limiting the amount of chemotherapy or radiation therapy prescribed. Administration of pure CSF-1 thus has the potential for increasing production of cells of the immune system, and consequently increasing the therapeutic to toxic ratio of anti-cancer drugs.
Of interest to the background of the invention is current research focused on hybridoma techniques for producing tumor cell lines which will manufacture highly specific monoclonal antibody to a selected antigenic substance. Techniques used for the production of monoclonal antibodies are generally well known in the art. A typical description of these procedures may be found in Oi, V. T. and L. A. Herzenberg, "Immunoglobulin Producing Hybrid", Mishell, B. B. and S. M. Shiigi (eds.), Selected Methods in Cellular Immunology, San Francisco: W. H. Freeman Publishing, 1979. Briefly summarized, lymphocytes removed from the spleen of an animal previously injected with the antigen of interest are induced to fuse with myeloma cells in the presence of polyethylene glycol. Thousands of "hybrid" myeloma cells are produced from the fusion. The supernatant from growth of each "hybridoma" cell culture is tested for the presence of the desired antibody activity. When such activity is found in the supernatant of one cell culture, it is cloned by limiting dilutions, and the clones produced are individually assayed for supernatant activity.
Due to the highly specific nature of their immunological properties, monoclonal antibodies developed according to hybridoma techniques have been proposed for use as diagnostic reagents, therapeutic agents, and agents for affinity purification of specifically cross-reactive antigenic proteins from crude sources. See, e.g., Chisholm, High Technology, 3, pp. 57-63 (1983) and U.S. Pat. Nos. 4,172,124 and 4,196,265.
While there exists a substantial need for specific monoclonal anti-CSF-1 antibodies for use in obtaining useful quantities of pure CSF-1 to advance on-going research concerning hematopoiesis and for potential use in treatment of immune cell deficiencies in patients undergoing anti-cancer treatments, there have been no reports of the successful use of hybridoma techniques in obtaining a monoclonal antibody to CSF-1.